Bordetella pertussis, the causative agent of the acute childhood respiratory disease whooping cough, is a human-adapted variant of Bordetella bronchiseptica, which displays a broad host range and typically causes chronic, asymptomatic infections. These pathogens express a similar but not identical surface-exposed and secreted protein called filamentous hemagglutinin (FHA) that has been proposed to function as both a primary adhesin and an immunomodulator. To test the hypothesis that FHA plays an important role in determining host specificity and͞or the propensity to cause acute versus chronic disease, we constructed a B. bronchiseptica strain expressing FHA from B. pertussis (FHA Bp) and compared it with wild-type B. bronchiseptica in several naturalhost infection models. FHA Bp was able to substitute for FHA from B. bronchiseptica (FHA Bb) with regard to its ability to mediate adherence to several epithelial and macrophage-like cell lines in vitro, but it was unable to substitute for FHA Bb in vivo. Specifically, FHA Bb, but not FHABp, allowed B. bronchiseptica to colonize the lower respiratory tracts of rats, to modulate the inflammatory response in the lungs of immunocompetent mice, resulting in decreased lung damage and increased bacterial persistence, to induce a robust anti-Bordetella antibody response in these immunocompetent mice, and to overcome innate immunity and cause a lethal infection in immunodeficient mice. These results indicate a critical role for FHA in B. bronchiseptica-mediated immunomodulation, and they suggest a role for FHA in host specificity.inflammation ͉ adhesin ͉ respiratory infection ͉ filamentous hemagglutinin D espite widespread vaccine coverage, whooping cough, or pertussis, remains a serious threat to human health, and its incidence has been increasing in recent years (1, 2). The causative agents, Bordetella pertussis and Bordetella parapertussis hu , are human-adapted pathogens that belong to a clade of very closely related Gram-negative bacteria that cause respiratory infections in mammals. Phylogenetic analyses indicate that Bordetella bronchiseptica, which displays a broad host range and typically colonizes its hosts chronically and asymptomatically, was the progenitor of this clade, with B. pertussis diverging relatively early and B. parapertussis hu diverging independently and much more recently than B. pertussis (3-6). Adaptation to humans and the propensity to cause acute disease (in which the infection is eventually cleared) rather than chronic disease (characterized by persistence of the bacteria, often for the lifetime of the host) has, therefore, evolved twice within this group of bacteria. Although a variety of Bordetella virulence factors have been characterized (4, 7-10), the mechanisms that determine host specificity and disease characteristics are not understood.Filamentous hemagglutinin (FHA), a primary component of acellular pertussis vaccines, is a large, -helical, highly immunogenic protein that is both surface-associated and secreted (11)(12)(13). In vitro...
Pertactin (PRN) is an autotransporter protein produced by all members of the Bordetella bronchiseptica cluster, which includes B. pertussis, B. parapertussis, and B. bronchiseptica. It is a primary component of acellular pertussis vaccines, and anti-PRN antibody titers correlate with protection. In vitro studies have suggested that PRN functions as an adhesin and that an RGD motif located in the center of the passenger domain is important for this function. Two regions of PRN that contain sequence repeats (region 1 [R1] and R2) show polymorphisms among strains and have been implicated in vaccine-driven evolution. We investigated the role of PRN in pathogenesis using B. bronchiseptica and natural-host animal models. A ⌬prn mutant did not differ from wild-type B. bronchiseptica in its ability to adhere to epithelial and macrophage-like cells in vitro or to establish respiratory infection in rats but was cleared much faster than wild-type bacteria in a mouse lung inflammation model. Unlike wild-type B. bronchiseptica, the ⌬prn mutant was unable to cause a lethal infection in SCID-Bg mice, but, like wild-type bacteria, it was lethal for neutropenic mice. These results suggest that PRN plays a critical role in allowing Bordetella to resist neutrophil-mediated clearance. Mutants producing PRN proteins in which the RGD motif was replaced with RGE or in which R1 and R2 were deleted were indistinguishable from wild-type bacteria in all assays, suggesting that these sequences do not contribute to PRN function.Bordetella pertussis and Bordetella bronchiseptica cause respiratory infections in mammals. These two closely related bacterial subspecies display a high level of biochemical and genomic similarity and produce a similar set of virulence factors that are regulated at the transcriptional level by a highly conserved two-component signaling system called BvgAS (8,46,50,65). However, they differ in symptomatic manifestations and host ranges: B. pertussis infects only humans and causes the acute disease whooping cough, while B. bronchiseptica has been isolated from nearly all mammals and typically causes chronic asymptomatic infections (41, 52). Nevertheless, some crucial factors are functionally interchangeable, such as filamentous hemagglutinin (FHA) (30) and the BvgAS signaling system (40), suggesting that studying B. bronchiseptica can reveal insights into the function of B. pertussis factors and vice versa. B. bronchiseptica and its natural-host animal models are therefore valuable tools for exploring molecular mechanisms of Bordetella pathogenesis.Pertactin (PRN) is a putative Bordetella virulence factor belonging to the autotransporter (AT) family. It was discovered as a surface-localized immunogen of B. bronchiseptica in 1985 (49) and was found to have highly conserved homologs in B. pertussis and B. parapertussis shortly thereafter (5, 38). Many subsequent studies focused on the immunogenic and protective properties of PRN, leading to its inclusion in acellular pertussis vaccines (34, 48), and efficacy trials have...
Summary Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human-adapted (e.g. Bordetella pertussis) strains produce a surface-exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg-Gly-Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural-host animal models should apply to B. pertussis FHA as well. We also show that the C-terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage-like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.
Purpose The therapeutic effect of trastuzumab monoclonal antibody (mAb) therapy has been shown to be partially dependent on functional NK cells. Novel agents that enhance NK cell function could potentially improve the anti-tumor effect of trastuzumab. We recently identified polysaccharide krestin (PSK), a natural product extracted from medicinal mushroom Trametes Versicolor, as a potent TLR2 agonist. The current study was undertaken to evaluate the effect of PSK on human NK cells and the potential of using PSK to enhance HER2-targeted mAb therapy. Experimental Design Human PBMC were stimulated with PSK to evaluate the effect of PSK on NK cell activation, IFN-γ production, cytotoxicity, and trastuzumab-mediated ADCC. Whether the effect of PSK on NK cells is direct or indirect was also investigated. Then in vivo experiment in neu transgenic mice was carried out to determine the potential of using PSK to augment the anti-tumor effect of HER2-targeted mAb therapy. Results PSK activated human NK cells to produce IFN-γ and to lyse K562 target cells. PSK also enhanced trastuzumab-mediated ADCC against SKBR3 and MDA-MB-231 breast cancer cells. Both direct and IL-12-dependent indirect effects seem to be involved in the effect of PSK on NK cells. Oral administration of PSK significantly potentiated the anti-tumor effect of anti-HER2/neu mAb therapy in neu-transgenic mice. Conclusion These results demonstrated that PSK activates human NK cells and potentiates trastuzumab-mediated ADCC. Concurrent treatment of PSK and trastuzumab may be a novel way to augment the anti-tumor effect of trastuzumab.
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